Despite recent advances in the care of critically ill patient, severe gram-negative infections remain a major cause of morbidity and mortality. Endotoxin (LPS) release from gram-negative bacteria is believed to produce many of the sequelae of sepsis. The macrophage is a major effector cell in the host's response to LPS and is pluritropic source of inflammatory mediators. Among the most important pathophysiologic mediators released in endotoxemia are the eicosanoid, prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT). Previous studies, including our own, using inhibitors of eicosanoid synthesis or specific receptor antagonists, have demonstrated pathogenic roles for the eicosanoids. This proposal seeks to delineate the cellular mechanism(s) of LPS stimulated eicosanoid synthesis and of the development of LPS tolerance. Two interrelated hypotheses to be tested are that: 1) altered guanine nucleotide regulatory (G) protein function contributes to LPS homologous desensitization (tolerance) and heterologous desensitization (cross-tolerance); and 2) de novo protein synthesis and protein kinase C activation are essential for LPS-induction of eicosanoid synthesis. The studies proposed to test the first hypothesis will characterize and identify specific G proteins transducing lipid A (the toxic moiety of LPS) stimulation of eicosanoids in rat peritoneal macrophages. The mechanism(s) of lipid A induced increases in macrophage membrane GTPase and of reduced GTPase activity during the development of LPS tolerance will be investigated. Studies will also investigate heterologous desensitization of TXA2 receptors in vascular tissue seen in LPS tolerance to determine if TXA2 receptor function, affinity and number, and/or coupling to second messenger systems are altered. The latter studies will ascertain if changes in cellular signal transduction in LPS tolerance extend to other tissues and receptors. Studies proposed in specific aims to test the second hypothesis will identify the potential eicosanoid regulatory proteins and synthesizing enzymes in lipid A stimulated eicosanoid production. Changes in macrophage production of the cytokines TNF, IL-1, and TGF-beta during the course of development of LPS tolerance will be assessed. The potential role of these cytokines in mediating or modulating lipid A-induced eicosanoid synthesis and changes in G protein function will be studied. The goal of this proposal is to delineate specific cellular mechanisms of; 1) LPS induced eicosanoid synthesis; and 2) homologous and heterologous desensitization that develop in LPS tolerance. Such data will provide new insights and potential novel therapeutic approaches for the treatment of endotoxemia and sepsis.